1. Field of the Invention
This invention relates to a cell-free process for producing cephalosporin antibiotics from peptides and derivatives thereof.
2. The Prior Art
The beta-lactam family of natural products includes the penicillins: ##STR2## cephalosporins: ##STR3## and cephamycins ##STR4## in which the beta-lactam ring is fused to a five or six membered sulfur-containing ring; together with clavulanic acid ##STR5## in which the beta-lactam is fused to a five membered oxygen-containing ring; the carbapenems ##STR6## in which the beta-lactam is fused to a five membered carbon containing ring; and the ##STR7## which are monocyclic compounds. Although there are many naturally occurring members of this family, only two can be used directly in medicine without structural change. These are penicillin G, the penicillin in which R=benzyl, and clavulanic acid. All other clinically important beta-lactam compounds have been prepared from one or other of the natural products by structural change. For many years the changes have been generally effected by substitution around the peripheries of the various ring systems and not in the ring systems themselves. Since 1974, however, efforts have been concentrated on nuclear modification of a beta-lactam natural product. Such efforts have generally resulted in complex chemical processes containing upwards of 16 steps with the result that the products are obtained in generally low yield and at extremely high cost. Moxalactam.RTM., for example, a third generation cephalosporin, is approximately five times more expensive than cephalothin, a first generation cephalosporin; and cephalothin is, in turn, approximately fifty times more expensive than ampicillin, a semi-synthetic penicillin (Drug Topics Red Book 1981).
Attention has therefore turned to alternative methods of synthesis, and in particular to microbiological methods. Cell-free syntheses of penicillins and the related cephalosporins are known in the art and attention is directed to U.S. Pat. No. 4,178,210 issued Dec. 11, 1979 to A. L. Demain et al, which teaches conversion only of the D-form, penicillin N, to a cephalosporin compound. In U.S. Pat. No. 4,248,966 issued Feb. 3, 1981, A. L. Demain et al teach the production of isopenicillin derivatives, in a cell-free system using an extract from Cephalosporium acremonium, from a tripeptide composed of unsubstituted or .beta. substituted D-valine, unsubstituted or substituted L-cysteine, and L-.alpha.-aminoadipic acid or its analogs. Freezing of the cell-free extract resulted in inactivation of certain enzymes so that conversion did not proceed past the isopenicillin stage. In U.S. Pat. No. 4,307,192 issued Dec. 22, 1981, A. L. Demain et al teach the use of a fresh (i.e. not frozen) cell-free extract of C. acremonium so as to preserve the racemase (epimerase) agent or agents necessary for the conversion of isopenicillin N to penicillin N, a necessary intermediate step in the process for conversion of L-aminoadipyl-L-cysteinyl-D-valine (abbreviated to LLD in the reference but hereinafter ACV) via an oxidative cyclization step to isopenicillin N, epimerization to penicillin N and oxidative ring expansion to desacetoxycephalosporin C. ##STR8## The activity of the racemase agent in a cell-free extract of C. acremonium was first recognized by Konomi et al, Biochem. J. Vol. 184, p 427-430, 1979, and confirmed by Baldwin et al, Biochem J. Vol. 194, 649-651, 1981, and Jayatilake et al, Biochem. J. Vol. 194, 649-647, 1981 who also recognized the extreme lability of the racemase agent so that recovery of the racemase agent per se is believed to be impossible. The lability of the racemase agent is believed to preclude use of cell-free extracts of C. acremonium for high yield commercial production of cephalosporins from peptide precursors.
Since about 1978, 6-aminopenicillanic acid has been produced commercially by the deacylation of benzyl penicillin using immobilized penicillin acylase (Proc. 1ST. European Congress of Biotechnology, Dechema Monographs, Volume 82, 162, 1978), and numerous other reactions have been suggested using immobilized biomaterials such as enzymes (Enzyme Engineering Vol. 6, 1982, Plenum).
It is, therefore, an object of the present invention to provide an integrated cell-free process for producing a cephalosporin compound from a peptide of the general formula ##STR9## where R.sub.1 is hydrogen, a lower alkyl or functionalized carboxylic group, and R.sub.2 is hydrogen or a lower alkyl group, using stable cell-free extracts from prokaryotic organisms.
It is another object of the present invention to provide immobilized cell-free extracts from prokaryotic organisms so as to permit continuous production of cephalosporins.
These and other objects of the invention will be apparent from the following description of the preferred embodiments.